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目的:检测肝癌干细胞标志CD133在化学诱导C57BL/6J小鼠肝癌过程中各时期的动态变化。方法:通过化学法[二乙基亚硝胺(diethylnirtosamine,DEN)/四氯化碳(carbon tetrachloride,CCl4)/乙醇]诱发50只C57BL/6J雄性小鼠肝癌,对照组为45只正常C57BL/6J雄性小鼠。观察小鼠成瘤情况和生长状态,对每2周定期处死小鼠获得的组织标本分别进行病理学切片观察,并采用实时荧光定量PCR、免疫组织化学法、蛋白质印迹法和FCM法分别检测CD133mRNA和蛋白的表达情况。结果:化学诱导20周后,成功诱发出小鼠肝癌。实时荧光定量PCR和FCM检测结果显示,第8周后诱癌组小鼠肝组织中CD133的表达高于正常对照组,差异有统计学意义(P<0.05或P<0.001);随着诱癌时间的增加,CD133的表达量呈上升趋势,第16周时诱癌组CD133的表达明显高于前期诱癌组(P<0.001)。蛋白质印迹法检测结果显示,诱癌组CD133蛋白从第4周起出现弱表达,随着诱癌时间的递增,CD133蛋白表达量逐渐增多。免疫组织化学检测结果显示,诱癌组第8周CD133出现弱阳性,第16周出现中等阳性,第20周出现强阳性;而正常对照组无表达。结论:肝癌干细胞标志CD133参与了肝癌的发生、发展全过程,随着诱癌进展其表达量逐渐增加。
OBJECTIVE: To detect the dynamic changes of liver cancer stem cell marker CD133 in various stages of chemically induced C57BL / 6J mice liver cancer. Methods: Fifty C57BL / 6J male mice were induced by chemical method [diethylnirtosamine (DEN) / carbon tetrachloride (CCl4) / ethanol]. The control group was 45 normal C57BL / 6J male mice. The tumorigenicity and growth status of the mice were observed. Tissue samples obtained from the mice sacrificed every 2 weeks were observed by pathological sections. The expression of CD133 mRNA was detected by real-time fluorescence quantitative PCR, immunohistochemistry, Western blotting and FCM And protein expression. Results: After 20 weeks of chemical induction, mouse liver cancer was successfully induced. The results of real-time quantitative PCR and FCM showed that the expression of CD133 in the liver tissue of cancer-inducing group after 8 weeks was higher than that of the normal control group (P <0.05 or P <0.001) With the increase of time, the expression of CD133 showed an upward trend. At the 16th week, the expression of CD133 in cancer-inducing group was significantly higher than that in the pre-cancerous group (P <0.001). The results of Western blotting showed that CD133 protein in cancer-inducing group was weakly expressed from week 4, and the expression of CD133 protein gradually increased with the increase of cancer-inducing time. The result of immunohistochemistry showed that CD133 appeared weakly positive on the 8th week in the cancer induction group, moderately positive on the 16th week, strong positive on the 20th week, but not expressed in the normal control group. Conclusion: CD133, a marker of hepatocellular carcinoma stem cells, is involved in the occurrence and development of hepatocellular carcinoma. The expression of CD133 gradually increases with the progress of carcinogenesis.